OBJECTIVETo establish and assess the Caco-2 cell in vitro absorption model.

METHODSCaco-2 cells were cultured on the millipore filters fixed in Snapwell transport chamber. The cell morphology, transepithelial electrical resistance, mannitol efflux rate and alkaline phosphatase activities were monitored during culture.

RESULTSAfter 21 days of in vitro culture, formation of tight junction was observed between the cells. The transepithelial electrical resistance reach a relatively stable value of 620-/+47 Omega.cm(2), the mannitol efflux rate was lower than 0.3%.h(-1).cm(-2), and the alkaline phosphatase activity in the apical side was significantly higher than that in the basolateral side.

CONCLUSIONThe established Caco-2 cell model shows similar morphology to intestinal epithelial cells with formation of polarity, and can be used as an in vitro model for absorption studies.

Caco-2 Cells ; Cell Culture Techniques ; methods ; Epithelial Cells ; cytology ; metabolism ; Humans ; Intestinal Absorption ; Intestines ; cytology

Animals ; Enteric Nervous System ; cytology ; Gastrointestinal Diseases ; etiology ; Gastrointestinal Motility ; physiology ; Humans ; Intestinal Mucosa ; cytology ; Intestines ; cytology ; innervation ; Muscle, Smooth ; cytology ; physiology ; Myenteric Plexus ; cytology ; Proto-Oncogene Proteins c-kit ; analysis

OBJECTIVETo investigate the injuries of intestinal mitochondria induced by different doses of whole-body radiation in Tibet minipigs.

METHODSEighteen Tibet minipigs were randomized into 5 radiation groups (n=3) and a control group (n=3). The minipigs in the radiation groups were subject to a total body X-ray radiation at 2, 5, 8, 11, or 14 Gy, and 72 h after the exposure, the mRNA expressions of the intestinal mitochondrial genes were examined using RT-PCR. The changes in the respiratory chain complexes I-IV and the respiratory functions of succinate and NADH were assayed, and the intestinal ultrastructures were observed using transmission electron microscopy (TEM) following the exposures.

RESULTSCompared with those in the control group, the expression levels of the related mitochondrial genes, the activities of the respiratory chain complexes and the function of the respiratory chain were significantly lowered in the radiation groups. At the doses below 8 Gy, the exposures caused significant reduction in the measurements as the radiation doses increased, but at higher doses, these measurements showed no further reductions. Ultrastructurally, exposures at 2 and 5 Gy caused mitochondrial expansion and mild reduction of the density, whereas radiation at 8 Gy or greater resulted in vacuolar changes and obvious expansion of the mitochondria with damages of the mitochondrial cristae and membranes.

CONCLUSIONBelow the doses of 8 Gy, intestinal mitochondrial damages in the minipigs increase with the radiation dose, but at higher doses, the damages do not further increase with the radiation dose.

Animals ; Dose-Response Relationship, Radiation ; Intestines ; cytology ; radiation effects ; Male ; Mitochondria ; radiation effects ; Radiation Dosage ; Swine ; Swine, Miniature

Animals ; Intestines ; cytology ; drug effects ; physiology ; Meperidine ; pharmacology ; Mice ; Mice, Inbred Strains ; Muscle, Smooth ; drug effects ; physiology ; Rabbits

OBJECTIVETo observe the effects of Dachengqi Decoction (大承气汤, DCQD) on morphological changes in the network of enteric nerve-interstitial cells of Cajal (ICCs)-smooth muscle cells (SMC) of enteric deep muscular plexuses (DMP) in the rats with multiple organ dysfunction syndrome (MODS).

METHODSOne hundred Wistar rats of both sexes weighing 200 to 250 g were randomly divided into the control group, MODS group, and DCQD group. The morphologic changes of enteric nerve-ICC-SMC network in the DMP of intestine was observed using c-Kit and vesicular acetylcholine transporter/neuronal nitric oxide synthase immunohistochemical double-staining with whole-mount preparation technique, confocal laser scanning microscopy, and electron microscopy.

RESULTSCompared with the control group, the distribution and densities of cholinergic/nitrergic nerves and ICC in the DMP (ICC-DMP) of intestine in the MODS group were significantly decreased (P<0.01), and the network of cholinergic nerve-ICC-SMC was disrupted; and the ultrastructural features of ICC-DMP, enteric nerve, and SMC were severely damaged. After treatment with DCQD, the damage in the network of enteric nerve-ICC-SMC was significantly recovered. Compared with the MODS group, the distribution and densities of cholinergic/nitrergic nerves and ICC-DMP in the DCQD group were significantly increased (P<0.01); and the ultrastructural features of ICC-DMP, enteric nerve, smooth muscle cells were significantly recovered.

CONCLUSIONSDCQD can improve the gastrointestinal motility in MODS. The mechanism may be related to the effect of repairing the damages in the network of enteric nerve-ICC-SMC.

Animals ; Interstitial Cells of Cajal ; cytology ; Intestines ; innervation ; Microscopy, Confocal ; Multiple Organ Failure ; physiopathology ; Plant Extracts ; therapeutic use ; Rats

OBJECTIVETo investigate the expression of R-spondin1 (RSpo1) in the intestinal epithelium of mice with intestinal ischemia-reperfusion injury and explore its significance.

METHODSFifty normal male Kunming mice were randomized into sham-operated group (n=10) and intestinal ischemia-reperfusion injury group (n=40), and in the latter group, the mice were subjected to 20-min intestinal mesenteric artery occlusion followed by reperfusion for 6, 12, 24, or 48 h. Enzyme-linked immunosorbent assay (ELISA) and RT-PCR were used to detect intestinal RSpo1 expression of the mice.

RESULTSThe results of RT-PCR and ELISA showed that RSpo1 expression was significantly decreased in mice at 6 h of reperfusion following the intestinal ischemia (P<0.05), and increased gradually with prolonged repersuion time, reaching the peak level at 24 h (P<0.05). The expression underwent rapid decrease afterwards to a significantly lower level than that in the control group at 48 h (P<0.05).

CONCLUSIONIntestinal ischemia-reperfusion injury may inhibit expression of RSpo1 in the early stage, and enhance its expression in the middle stage. RSpo1 can promote proliferation and differentiation of intestinal epithelial stem cells and plays an important role in the repair intestinal mucosal damage.

Animals ; Cell Proliferation ; Intestinal Mucosa ; cytology ; metabolism ; Intestines ; blood supply ; metabolism ; Male ; Mice ; Random Allocation ; Reperfusion Injury ; metabolism ; Stem Cells ; cytology ; Thrombospondins ; genetics ; metabolism

OBJECTIVETo study the absorption of 3-epidehydrotumulosic acid (EDHTA), polyporenic acid C (PPAC) and 6alpha-hydroxypolyporenic acid C (HPPA) isolated from the sclerotium of Poria cocos. In human intestinal epithelial.

METHODBy using Caco-2 (the human colonic adenocarcinoma cell lines) cells monolayer as an intestinal epithelial cell model, the permeability of EDHTA, PPAC and HPPA were studied from apical side (AP side) to basolateral side (BL side) or from BL side to AP side. The three compounds were measured by reversed-phase high performance liquid chromatography (HPLC) coupled with UV detector. Transport parameters and apparent permeability coefficients (P(app)) were then calculated and compared with those of propranolol and atenolol, which are the transcellular transport marker and as a control substance for high and poor permeability, respectively.

RESULTThe P(app) values of EDHTA, PPAC and HPPA were (9.99 +/- 1.08) x 10(-6), (10.06 +/- 0.53) x 10(-6) and (3.32 +/- 0.66) x 10(-6) cm x s(-1) from AP side to BL side, and (17.76 +/- 0.91) x 10(-6), (19.23 +/- 1.16) x 10(-6) and (8.19 +/- 0.57) x 10(-6) cm x s(-1) from BL side to AP side,respectively. Under the condition of this experiment, the P(app) value was 1.45 x 10(-5) cm x s(-1) for propranolol and 4.22 x 0(-7) cm x s(-1) for atenolol. The P(app) values of EDHTA and PPAC were at a nearly same magnitude with those of propranolol, and HPPA lied between those of propranolol and atenolol. On the other hand, the efflux transport of EDHTA, PPAC and HPPA were higher 1.78, 1.91 and 2. 47 times more than its influx transport with 0.56, 0.52 and 0.41 rate of P(app A --> B)/P(app (B --> A).

CONCLUSIONEDHTA, PPAC and HPPA can be absorbed across intestinal epithelial cells, among EDHTA and PPAC will be completely. HPPA will be moderately absorbed compounds. EDHTA, PPAC and HPPA may have been involved in efflux mechanism in Caco-2 cells monolayers model from the basolateral-to-apical direction.

Biological Transport ; Caco-2 Cells ; Chromatography, High Pressure Liquid ; Epithelial Cells ; cytology ; metabolism ; Humans ; Intestinal Absorption ; Intestines ; cytology ; Linear Models ; Permeability ; Poria ; chemistry ; Triterpenes ; metabolism ; pharmacokinetics

OBJECTIVETo study the effects of licorice on the proliferation of intestinal crypt stem cell line IEC-6 and the expression of p53.

METHODSInduced by difluoro-methylornithine (DFMO), polyamine-depleted IEC-6 cells under growth inhibition were used as the pathological cell model in this study. Cells were divided into four groups, i. e., the control group, the DFMO-treated group, the high dose licorice group, and the low dose licorice group. The control group consisted of IEC-6 cells cultured in normal condition. The other three groups were all treated with 5 mmol/L DFMO. The high dose and low dose licorice groups were supplemented with 40 and 80 microg/mL licorice granule respectively. All the groups were cultured for 6 successive days. The cell number and viability were determined using flow cytometry. The level of p53 protein was detected by Western blot. The p53 mRNA levels and stability were detected using fluorescent quantitative Real-time PCR.

RESULTSCompared with the control group, the cell growth of the DFMO group was obviously inhibited on the 4th day (P < 0.05). The cell number increased more obviously in the low dose licorice and the high dose licorice groups in a dose-dependent way on the 6th day when compared with the DFMO group (P < 0.05). When compared with the control group, significantly elevated expression levels of p53 protein and mRNA in cells of the DFMO group were detected after 6-day treatment (P < 0.05). When compared with the DFMO group, the expression levels of p53 protein and mRNA were significantly down-regulated in the low dose licorice and the high dose licorice groups (P < 0.05). The degradation of p53 mRNA was the fastest in the control group, while the degradation speed of cells in the DFMO group was the slowest.

CONCLUSIONOne of mechanisms for protective and healing effects of licorice on the intestinal mucosa was possibly through down-regulating the stability of p53 mRNA, lowering the expression of p53, thus promoting the proliferation of the intestinal crypt stem cells.

Animals ; Cell Line ; Cell Proliferation ; Glycyrrhiza ; Intestines ; cytology ; metabolism ; RNA Stability ; drug effects ; RNA, Messenger ; genetics ; Rats ; Stem Cells ; cytology ; drug effects ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo investigate the dynamic changes of intestinal epithelial stem cells during the injured-repaired progress induced by 5-FU.

METHODSFifty adult C57BL/6J mice were enrolled in this study, 40 of them were intraperitoneally injected with 5-FU (30 mg per kg of body weigh) for five days, and 10 of them intraperitoneally injected with PBS as control. At day 1, 3, 5, 7 after treatment, the mice were killed and middle intestine was taken. Pathology was examined by HE staining. Musashi-1 (msi-1) expression was detected by immunohistochemical technique. The percentage of Rho low staining cells was detected by flow cytometry.

RESULTSAfter treatment with 5-FU, the intestinal mucosa was damaged. The Rho low staining cells were increasing, and at day 1 after treatment, the percentage of Rho low staining cells reached the highest level (P<0.01). The number of cells expressing msi-1 did not change significantly (P>0.05), but the percentage of positive msi-1 cells increased significantly (P<0.01). There was positive correlation between the percentage of Rhodamine 123 low staining cells and positive msi-1 cells in each group (r=0.867, P<0.01).

CONCLUSIONSThe Rho low staining cells may contain rich intestinal epithelial stem cells. The intestinal epithelial stem cells expressing msi-1 can regenerate the damage of intestinal mucosa induced by 5-FU.

Animals ; Cell Line ; Epithelial Cells ; cytology ; drug effects ; Female ; Fluorouracil ; adverse effects ; Intestinal Mucosa ; cytology ; drug effects ; pathology ; Intestine, Small ; cytology ; drug effects ; pathology ; Intestines ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Stem Cells ; drug effects

OBJECTIVETo explore the effects of gastrin on the expression of 1,25(OH)2D3-membrane associated rapid response steroid (1,25D3-MARRS) binding protein in rat intestinal epithelium.

METHODSSD rats received intraperitoneal injections of gastrin, omeprazole or physiological saline. The protein expression of 1,25D3-MARRS binding protein in SD rat intestinal was determined with Western blotting and immunohistochemistry, and its mRNA levels determined by RT-PCR. The serum calcium and phosphate levels in the rats were also detected.

RESULTSImmunohistochemistry showed that 1,25D3-MARRS binding protein was expressed mainly in the nuclei, cytoplasm and membrane of the intestinal epithelial cells. Both the protein and mRNA expression levels of 1,25D3-MARRS binding protein were up-regulated after treatments with gastrin and omeprazole (P<0.05), but the serum calcium and phosphate concentrations showed no obvious increase.

CONCLUSION1,25D3-MARRS binding protein, which is widely expressed with versatile functionalities, is regulated by gastrin and shows high potentials in the study of gastrointestinal diseases.

Animals ; Calcitriol ; metabolism ; Epithelial Cells ; drug effects ; metabolism ; Gastrins ; pharmacology ; Intestines ; cytology ; drug effects ; Male ; Protein Disulfide-Isomerases ; metabolism ; Rats ; Rats, Sprague-Dawley